1. Technical Field
The present invention relates to the virtual detection, quantization and characterization of immunologically detected substances electronically in human and animal biological fluids such as whole blood, serum, plasma, urine, milk, pleural and peritoneal fluids, and semen, which detection, quantification and characterization is performed in a thin chamber on a quiescent fluid sample, said chamber having at least two parallel planar walls, at least one of which is transparent.
2. Background Information
This invention relates to the improvement in the performance of all immunoassays that presently involve the physical separation of bound from free analyte by, instead, performing a virtual separation of bound and free optically detected label in a ligand assay, wherein the label is preferably a fluorescent label although any optically detectable and quantifiable label will suffice. The chambers for use in this assay and the instruments for measuring the analytes in these chambers are described in the following issued U.S. Pat. Nos. 6,929,953 issued to S. C. Wardlaw; 6,869,570 issued to S. C. Wardlaw; 6,866,823 issued to S. C. Wardlaw; and U.S. Patent Application Publication No. US 2007/0087442, to S. C. Wardlaw, published Apr. 19, 2007.
Physical separation of bound from free analytes have, in the prior art, been accomplished by multiple means including but not limited to, adsorption of the free label by charcoal or talc, magnetic separation of beads containing either the bound or unbound analyte, adsorption of the bound labeled analyte by the container such as antibodies coupled to the wall of a test tube and the use of second precipitation antibodies directed against the analyte binding antibody followed by centrifugation as well as the methods described in the above noted patents and publications.
Some of the types of prior art physical separation of bound target analyte assays are described in the following U.S. Pat. Nos. 5,834,217; 5,776,710; 5,759,794; 5,635,362; 5,593,848; 5,342,790; 5,460,979; 5,480,778; and 5,360,719, all issued to R. A. Levine et al. In the aforementioned patents, the separation of bound from free analyte is performed by centrifugation, or other physical methods, such as decanting, filtration, or the like.
The prior art also describes a type of immunoassay, which is called a “homogeneous immunoassay”. Homogeneous immunoassays do not require the physical separation of bound from non-bound, or free, analyte. The “separation of the bound from free” is accomplished by utilizing the steric interference of an enzyme by the relatively large antibody and quantifying the colored or fluorescent products of the enzymatic action. Additional methods of homogeneous assays utilize the fluorescent quenching of fluorophores to distinguish bound from free analyte. While these methods greatly simplify the performance of immunoassays, they are generally useful only for high concentrations of analytes with low molecular weight since the large molecular weight of target analytes such as proteins (e.g., insulin), growth hormones, and the like will also interfere with the enzyme and may affect quenching. Additionally immunoassays of this homogeneous type typically do not have the high sensitivity of standard immunoassays.
It would be highly desirable to provide a ligand assay of a target analyte wherein the quantification of the target analyte is a virtual one which can be performed electronically thereby having the advantages of a homogeneous immunoassay while maintaining the sensitivity of standard immunoassays, as well as the ability to have large size target analytes such as hormones like insulin, growth hormone and the like.